The goal of this project is investigate pathophysiological basis of high protein intake-induced hypercalciuria. Epithelial Ca 2+ channels (ECaC1) are present in the apical membrane of the distal convoluted tubule (DCT) of kidney and play an important role in the maintenance of overall calcium homeostasis. Our preliminary results indicate that hypercalciuria associated with a high protein intake is, at least partly, caused by acid inhibition of ECaC1-mediated Ca 2+ reabsorption in DCT. In Specific Aim 1, we will examine the molecular mechanism of acid inhibition of ECaC1. Likely candidates of "pH sensor" for acid regulation of ECaC1 will be mutated by site-directed mutagenesis. The activity of wild type and mutant channels will be examined by whole-cell patch-clamp recording. Membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2) has recently emerged as a unique regulator of channel function. In Specific Aim 2, we will examine the hypothesis that PIP2 regulation of ECaC1 channel underlines the mechanism by which prostaglandin E2 worsens the high protein intake-induced hypercalciuria. In Specific Aim 3, we will confirm that luminal acidification inhibits Ca 2+ reabsorption in the distal convoluted tubules (DCT). In vivo microperfusion of rat DCT will be performed to examine the effect of luminal pH on Ca 2+ reabsorption. These studies will help understand the mechanism of hypercalciuria, not only during high dietary protein intake but also under conditions of chronic metabolic acidosis.